The primary objective of Project 1 is to define the mechanisms of functional impairment of HCV-specific CD8 T cells associated with viral persistence. While viral escape mutations within T cell epitopes occur in HCV immune evasion, a significant proportion of CD8 T cells in chronically infected subjects recognize HCV epitopes that do not demonstrate escape mutations. It is thus likely that functional impairment of CD8 cells specific for non-escaped epitopes is an additional important factor in HCV persistence. We propose that a comprehensive comparative analysis of HCV-specific CD8 T cell phenotype and function among patients who clear HCV infection versus those who do not, and between T cells recognizing epitopes that undergo substitution and those that do not will reveal specific molecular and cellular mechanisms of T cell unresponsiveness relevant to viral persistence. Specifically, we aim 1)To perform a set of in vitro functional analyses assessing the capacity of HCV specific CD8 T cells of various previously characterized phenotypes to produce relevant effector cytokines and to perform killer functions and 2)To develop and characterize an in vivo cytotoxic lymphocyte (CTL) assay for functional analysis of tetramer+ HCV specific CD8 cells using adoptive transfer into an established NOD/SCID/--/- system. This will allow us to directly analyze the in vivo functional effects of antibodies and/or cytokines targeted at potentially relevant cell membrane receptors on human HCV specific CD8 T cells. Using specific antagonist antibodies, candidate molecular determinants of CD8 T cell unresponsiveness will be interrogated in this novel in vivo system in which human T cells from patients are adoptively transferred into receptive immunodeficient mice. Outcomes of this interrogation will have direct translational relevance to the immunotherapy of chronic HCV infection as well as enhancing understanding of T cell impairment associated with persistent infection. Results of studies of humoral immune responses from Project 2 will be integrated to expand knowledge of the interplay between humoral and cellular immune responses to HCV in humans. We have already demonstrated that we can obtain the critical specimens required for this investigation and will be able to conduct unique longitudinal studies of adaptive immune responses in acute HCV.